Diminished DEFA6 Expression in Paneth Cells Is Associated with Necrotizing Enterocolitis

Hindawi Gastroenterology Research and Practice Volume 2018, Article ID 7345426, 7 pages https://doi.org/10.1155/2018/7345426

Research Article Diminished DEFA6 Expression in Paneth Cells Is Associated with Necrotizing Enterocolitis

1 2 3 1 Laszlo Markasz , Alkwin Wanders, Laszlo Szekely, and Helene Engstrand Lilja

1Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden 2Department of Biomedical Sciences, Umeå University, Umeå, Sweden 3Department of Laboratory Medicine, Division of Pathology, Karolinska Institute, Stockholm, Sweden

Correspondence should be addressed to Laszlo Markasz; [email protected]

Received 5 July 2018; Accepted 28 August 2018; Published 21 October 2018

Academic Editor: Stephen Fink

Copyright © 2018 Laszlo Markasz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Necrotizing enterocolitis (NEC) is the most common gastrointestinal disorder in premature infants with a high morbidity and mortality. Paneth cell dysfunction has been suggested to be involved in the pathogenesis of NEC. Defensin alpha- 6 (DEFA6) is a specific marker for Paneth cells acting as part of the innate immunity in the human intestines. The aim of this study was to investigate the expression of DEFA6 in infants with NEC. Materials and Methods. Infants who underwent bowel resection for NEC at level III NICU in Sweden between August 2004 and September 2013 were eligible for the study. Macroscopically vital tissues were selected for histopathological evaluation. All infants in the control group underwent laparotomy and had ileostomy due to dysmotility, and samples were taken from the site of the stoma. DEFA6 expression was studied by immunohistochemistry. Digital image analysis was used for an objective and precise description of the samples. Results. A total of 12 infants were included in the study, eight with NEC and four controls. The tissue samples were taken from the colon (n =1), jejunum (n =1), and ileum (n =10). Both the NEC and control groups consisted of extremely premature and term infants (control group: 25–40 gestational weeks, NEC group: 23–39 gestational weeks). The postnatal age at the time of surgery varied in both groups (control group: 4–47 days, NEC group: 4–50 days). DEFA6 expression in the NEC group was significantly lower than that in the control group and did not correlate with gestational age. Conclusion. The diminished DEFA6 expression in Paneth cells associated with NEC in this study supports the hypothesis that alpha-defensins are involved in the pathophysiology of NEC. Future studies are needed to elucidate the role of alpha-defensins in NEC aiming at finding preventive and therapeutic strategies against NEC.

1. Introduction intestinal ischemia, and colonization with pathogenic bacteria [1, 4]. The condition is an inflammation in the intestines Necrotizing enterocolitis (NEC) is a serious gastrointestinal related to a harmful overreaction of the immature immune disorder that affects 10–15 percent of premature infants with system to some insults [4]. The histological appearance a birth weight under 1500 g [1]. The mortality rates are up to shows bacterial invasion of the epithelia and early signs of 50% for infants requiring surgery [2]. Morbidity includes necrosis of the enterocytes at the top of some villi [5]. poor neurodevelopmental outcome and short bowel syn- Paneth cells are specialized epithelia that play a major drome [3]. role in the innate immune response [6]. They protect intes- Current evidence suggests a multifactorial cause of NEC tinal stem cells from pathogens, stimulate stem cell differen- [1]. Prematurity is the main risk factor, presumably due to tiation, and assist in repairing the intestine. The stem cell immaturity of gastrointestinal motility, intestinal barrier compartment is located at the base of the crypts, in the small function, and immune defence. Other contributing factors intestine interspersed by Paneth cells [7]. Paneth cells act are thought to be genetic predisposition, enteral feeding, through the production of antimicrobial proteins/peptides 2 Gastroenterology Research and Practice

[8]. The principal antimicrobial peptides, the alpha- ganglion cells. The fourth control was later diagnosed with defensins DEFA5 and DEFA6, differ in function [9]. DEFA6 pseudoobstruction and has still an ileostomy. shows weak activity against bacteria [10], and its antimicrobial function is activated by reducing milieu as a one- 2.2. Intestinal Tissue Samples and Immunohistochemistry. step mechanism by getting the more hydrophobic [11]. We performed both the immunohistochemistry and the DEFA6 may have a key role in protecting the small intes- image analysis blindly. All samples were sectioned and tine against invasion by diverse enteric pathogens through stained on the same occasion for comparable analysis. Intes- self-assembled peptide nanonets [12]. tinal tissues were fixed in 4% formaldehyde in PBS for 24 h at DEFA5 and DEFA6 mRNA levels are detectable at 13.5– 4°C. Embedded samples were sectioned (3 μm) and mounted 17 weeks of gestation in the small intestine but with markedly on SuperFrost slides. Samples were deparaffinized through a diminished expression until the middle of the third trimester graded series of xylol-ethanol. To determine the Paneth cell- [13]. Enteric DEFA5 and DEFA6 mRNA levels are signifi- specific expression of DEFA6, immunohistochemistry was cantly lower in fetus and term newborns than in adults, and performed by the Benchmark Ultra system (Ventana) and there are fewer numbers of Paneth cells in the crypts in the ultraView Universal DAB Detection Kit (Ventana) and extreme prematures than in term newborns and adults [13]. even counterstaining with hematoxylin-eosin was carried Recent studies suggest a key role for Paneth cells in the out. Tissue sections were incubated with a polyconal rabbit pathophysiology of NEC [5, 14–16]. The pathogenesis of anti-DEFA6 antibody (Prestige Antibodies® Powered by NEC and inflammatory bowel Crohn’s disease show many Atlas Antibodies, 1 : 800 dilution) after antigen retrieval in similarities [5, 17]. In Crohn’s, the production of both the CC2 buffer (Ventana) for 36 min. Negative control sec- DEFA5 and DEFA6 by Paneth cells is reduced [18]. Current tions were prepared by performing immunostaining proce- knowledge of alpha-defensins in NEC is scanty, and the dures without adding primary antibodies. Representative results seem to be controversial [19–21]. A better under- sections were digitally scanned with a 3DHISTECH scanner. standing of NEC is crucial to developing prevention and The images were exported in TIFF format (10x magnifica- treatment strategies. tion) with the virtual microscope software Panoramic Viewer The aim of the study was to investigate the expression of (3DHISTECH). DEFA6 in infants with NEC. 2.3. Image Analysis. ImageJ (freeware) was used for semiau- tomatic image analysis. The color detection of DAB staining 2. Materials and Methods was performed by the IHC Toolbox plugin in ImageJ, which can be effectively used to analyze samples stained by immu- 2.1. Study Population. Infants who underwent bowel resec- nohistochemistry [23]. The model for color detection of tion for NEC at level III NICU in Sweden between August brown pixels (which corresponded to the DAB staining and 2004 and September 2013 were eligible for the study the level of DEFA6 expression) was adjusted specifically for (Table 1). The study protocol was approved by the Regional the present project. The specificity of color detection was Ethical Review Board, and written informed consent was controlled visually. Working with image stacks during the obtained from the parents. evaluation process allowed the analysis to be made compara- Oral feeding with breast milk was started within two ble between images. After color detection of DAB staining, hours after birth in premature infants both in the control the RGB color images were converted to 8-bit files. Inversion and the NEC groups. NEC was diagnosed by radiological of the pixel intensity values resulted in higher pixel intensity and clinical features and staged according to the criteria of corresponding to higher DEFA6 expression. The workflow is Bell et al. [22]. NEC diagnosis was confirmed during surgery presented in Figures 1(a)–1(e). Before analysis, the same and by histopathological evaluation. Infants with NEC were threshold window was set on each image in order to filter treated with broad-spectrum antibiotics, and enteral feeding unspecific too high and/or too low pixel values. Besides the was ceased prior to surgery. Infants with NEC underwent advantages of the method, even some limitations could be bowel resection, and an enterostomy was created. Those considered: the antigen retrieval process and the staining samples that represented macroscopically vital tissue, from process are not easy to standardize and both can influence ends of the resected intestine, were selected for further histo- DAB intensity. Thus, the measured intensity levels in differ- pathological evaluation, and samples with complete mucosal ent studies are not directly comparable if they are performed erosion were excluded. at different time points. The controls all presented with delay to pass meconium, abdominal distention, and bilious vomiting, and they under- 2.4. DEFA6 Expression. Comparison of the general expres- went laparotomy between four and 47 days of life and an sion in the tissue was difficult since the height of the mucosa ileostomy was created. Samples from the controls used for showed variations between patients. A solution was to mea- staining were taken from the site of the stoma. Three of the sure the DAB-stained area as we hypothesized that the stain- controls had transient functional immaturity of the intestine, ing was homogeneous and had the same intensity for all the and they were successfully managed with a temporary ileost- samples. We generated an even more standardized process, omy (Table 1). Colonic biopsies in these three controls which showed DEFA6 expression for each μm length of the revealed the presence of immature ganglion cells, and ileal mucosa. The mucosal part of the sections was selected man- biopsies from the stoma showed the presence of normal ually as the region of interest (ROI) (Figure 1(b)), and the size Gastroenterology Research and Practice 3

Table 1: Study population.

Tissue Gestational age at birth Birth weight Gestational age at surgery Postnatal age at Patient code Diagnosis Gender origin (weeks + days) (grams) (weeks + days) surgery (days) 12244-04_B1 NEC Ileum 27 + 6 1438 28 + 4 5 Male 9589-10_B NEC Ileum 23 + 2 585 30 + 3 50 Male 12106-10_C NEC Ileum 26 + 2 1025 27 + 1 6 Male 18596-11_L NEC Colon 39 + 0 2360 39 + 4 4 Male 23543-11_B NEC Ileum 29 + 2 1225 29 + 6 4 Male 6126-12_A NEC Jejunum 26 + 2 912 31 + 0 33 Male 6436-13_C NEC Ileum 24 + 1 597 25 + 6 12 Female 33-04_B NEC Ileum 34 + 3 2300 35 + 2 6 Female 13749-07_A Dysmotility Ileum 36 + 4 3075 39 + 6 23 Male 3267-13 Dysmotility Ileum 25 + 6 622 31 + 1 37 Female 315-10_1 Dysmotility Ileum 40 + 4 4320 41 + 1 4 Female 873-12_5 Dysmotility Ileum 25 + 0 870 31 + 5 47 Female

(a) (b) (c) (d) (e)

Figure 1: Image processing: DAB staining detection. (a) Original image—RGB color. (b) Selection of the mucosa as the region of interest (ROI). (c) IHC Toolbox plugin in ImageJ selects color, representing DAB staining. (d) Conversion of an RGB image to an 8-bit image (the lowest pixel intensity represents the highest DEFA6 expression and vice versa). (e) Inversion of pixel intensity values results in the highest pixel intensity corresponding to the highest DEFA6 expression. of the area and the total DAB intensity of ROI were measured 3. Results and Discussion by ImageJ. 3.1. Study Population. A total of 12 infants were included in the study, eight with NEC and four controls. Patient charac- “ ” Length of mucosa in the ROI teristics are shown in Table 1. The tissue samples were taken Area of ROI from the colon (n =1), jejunum (n =1), and ileum (n =10). = , Mean height of mucosa Both the NEC and control groups consisted of extremely premature and term infants (control group: 25–40 gestational DEFA6 expression Total DAB intensity of ROI weeks, NEC group: 23–39 gestational weeks). The postnatal = μm “Length” of mucosa in the ROI μm age at the time of surgery varied in both groups (control – – 1 group: 4 47 days, NEC group: 4 50 days). 3.2. Tissue Characteristics and DEFA6 Expression. Searching We calculated the mean value of the mucosal height (μm) in the database of the Human Protein Atlas revealed that ﬁ for each section from ten representative sites measured by the DEFA6 is a highly speci c marker for Paneth cells with Panoramic Viewer software. Length in μm could be con- exclusive expression in the intestine in adults [24]. verted into pixels and vice versa. The “length” of the mucosa The height of the mucosa varied highly in patients inde- part in the ROI is calculated by dividing the area in pixels pendently of the origin of the tissue or the indication for with the height of the mucosa. operation. No correlation between the height of the mucosa and the level of DEFA6 expression or gestational age could 2.5. Statistics. Statistics were assessed in Microsoft Excel by be seen (data not shown). two-sample t-test. A P value of <0.05 was considered to be We found Paneth cells in all samples (Figure 2), even in statistically signiﬁcant. the colon tissue (Figure 2, F). The tissue samples showed 4 Gastroenterology Research and Practice

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

(a)

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

(b)

Figure 2: DEFA6 expression in the study material. (a) Increasing order of DEFA6 expression in patients. NEC group: A–F, H, and J; control group: G, I, K, L. (b) The lookup tables of images represent the distribution levels of DEFA6, visualized by colors (A–L). Extracellular DEFA6 expression appears in cases of higher general DEFA6 levels (H–L). different levels of DEFA6 expression (Figure 3). Tissue sam- independently of how we evaluated the DAB staining ples may have varying water content that can influence DAB (DEFA6 expression/μm mucosa (P =0019) or percent staining levels; therefore, we performed measurements on the area of the mucosa with DEFA6 expression (P =0003)) DAB-covered area as well. DEFA6 expression was signifi- (Figure 3). The DEFA6 expression in the NEC group cantly lower in the NEC group than in the control group, remained low and did not correlate with gestational age; Gastroenterology Research and Practice 5

12000 immunohistochemistry was not previously available. The DAB intensity/m mucosa use of immunohistochemistry in our study enabled struc- 10000 tural evaluations. The disadvantage of this method is that it detects the actual protein levels, which may not correlate 8000 with mRNA levels in case of circumstances with enhanced protein degradation. 6000 P: 0,019 Salzman et al. found elevated DEFA5 and DEFA6 mRNA levels in infants with NEC compared with five near-term 4000 controls (four patients with intestinal atresia and one with 2000 meconium ileus) [20]. Intracellular peptide levels in NEC did not coincide with the elevation in mRNA. Puiman 0 et al. found decreased DEFA5 protein expression in NEC NEC Control patients [21]. The control group consisted of various diagnoses, namely, intestinal atresia, volvulus, gastroschisis, cloacal malformation, milk curd obstruction, Meckel’s diverticulum, 50 and ileus. The comparison with results of other studies is 45 DAB area % complicated by methodological heterogeneity and variation 40 in gestational age between study populations and variation 35 in diagnosis in the control groups [20, 21]. Nevertheless, 30 similar to our results, alteration in the expression of alpha- P: 0,003 25 defensins DEFA5 and DEFA6 seems to be associated with NEC [20, 21]. 20 The number of Paneth cells [25] and the enteric expres- 15 sion of alpha-defensins in the fetus are low [13]; however, 10 the production after birth is inducible by multiple factors. 5 Both intraluminal bacteria and lipopolysaccharide can stim- 0 ulate Paneth cell secretion, as well as cholinergic agonists NEC Control and feeding [25–28]. This corresponds to our observation that DEFA6 expression was high in our fed extremely prema- Figure 3: DEFA6 expression in the NEC group and the control ture controls. An interpretation of our observations is that fi ff group. Signi cant di erences in DEFA6 expression irrespective of the DEFA6 expression is initially low at birth, inducible, the evaluating method. and increasing in healthy individuals but remains low in patients who develop NEC. however, higher DEFA6 expression appeared in premature Interestingly, we found Paneth cell metaplasia in a controls (Figure 4). colonic tissue sample with NEC. This phenomenon has been We were not able to estimate the objective count of previously reported in inflammatory bowel disease [29]. Pui- Paneth cells due to the confluence of DAB staining in the sec- man et al. described metaplastic Paneth cells in post-NEC tions with higher DEFA6 expression. However, the general stricture colon samples but not in NEC samples. They sug- impression suggests lower Paneth cell density in the NEC gested that chronic inflammation caused Paneth cell meta- group (Figure 2). plasia [21]. The strength of this study was that it was one of the two 3.3. Discussion. In the present study, we found a diminished studies in DEFA6 expression and NEC. Moreover, we DEFA6 expression in Paneth cells from infants with NEC described the setup of a reliable semiquantitative method to compared to controls. Paneth cells and their main products, study the expression of DEFA6. alpha-defensins, have been well studied in the development The limitations of our study were the small sample size of Crohn’s disease in adults [7, 16]. In Crohn’s, the produc- and the fact that the numbers of age-matched control tion of DEFA5 and DEFA6 by Paneth cells was reduced patients were limited since material collection from a healthy [15], which results in a defective antimicrobial shield and control group was not feasible due to ethical reasons. The dysfunction of the mucosal barrier [9]. In premature same problem appeared in previous studies of DEFA5 and infants, the diminished expression of DEFA5 and DEFA6 DEFA6 where patients with various diagnoses such as volvu- may result in higher risk of infection and inflammation. lus, gastroschisis, small intestinal atresia, and ileus were Although recent studies have suggested a Paneth cell included as controls [20, 21]. dysfunction in NEC [5, 11–13], studies in alpha-defensin DEFA5 and DEFA6 expression in NEC patients are 4. Conclusions restricted to two previous reports [20, 21]. They investi- gated the mRNA levels of DEFA5 and DEFA6 and protein The diminished DEFA6 expression in Paneth cells associated level of only DEFA5 but not DEFA6 [20, 21]. An explana- with NEC in this study supports the hypothesis that alpha- tion to the absence of studies in DEFA6 protein levels in defensins are involved in the pathophysiology of NEC. NEC might be that the reliable commercial antibody for Future studies are needed to elucidate the role of alpha- 6 Gastroenterology Research and Practice

12000 12000

10000 10000

8000 8000

6000 6000 R2 = 0.5007 R2 = 0.6407 4000 4000 R2 = 0.0019 DAB intensity/ m mucosa 2000 R2 = 0.068 DAB intensity/ m mucosa 2000

0 0 150 200 250 300 150 200 250 300 Gestational age (days) Postconceptional age at surgery (days) NEC NEC Control Control (a) (b)

Figure 4: Relationship between DEFA6 expression and age. (a) Control patients showed higher level of DEFA6 expression and negative correlation between DEFA6 expression and gestational age. Premature controls had the highest DEFA6 level. Most of the NEC patients showed low DEFA6 expression. No correlation could be confirmed between DEFA6 expression and gestational age in the NEC group. (b) The phenomenon above was more pronounced between DEFA6 expression and postconceptional age at surgery (maturation level). corr: 0.03 (NEC group), corr: −0.74 (control group). defensins in NEC aiming at finding preventive and therapeu- [5] S. J. McElroy, M. A. Underwood, and M. P. Sherman, “Paneth tic strategies against NEC. cells and necrotizing enterocolitis: a novel hypothesis for disease pathogenesis,” Neonatology, vol. 103, no. 1, pp. 10–20, 2013. Data Availability [6] J. Paneth, “About the secreting cells in the small intestinal epi- ” – The data used to support the findings of this study are thelium, Arch Mikroskop Anat, vol. 31, pp. 113 191, 1888. “ fi included within the article. [7] N. Barker, J. H. van Es, J. Kuipers et al., Identi cation of stem cells in small intestine and colon by marker gene Lgr5,” Nature, vol. 449, no. 7165, pp. 1003–1007, 2007. Conflicts of Interest [8] A. J. Ouellette, “Defensin-mediated innate immunity in the ” fl small intestine, Best Practice & Research Clinical Gastroenter- The authors declare that they have no con icts of interest. ology, vol. 18, no. 2, pp. 405–419, 2004. [9] P. Chairatana, H. Chu, P. A. Castillo, B. Shen, C. L. Bevins, and Acknowledgments E. M. Nolan, “Proteolysis triggers self-assembly and unmasks innate immune function of a human α-defensin peptide,” This work was supported by funding from H.R.H. Crown Chemical Science, vol. 7, no. 3, pp. 1738–1752, 2016. Princess Lovisa’s Association for Paediatric Health Care/ [10] B. Ericksen, Z. Wu, W. Lu, and R. I. Lehrer, “Antibacterial Foundation Axel Tielman’s Memorial Fund. activity and specificity of the six human α-defensins,” Antimi- crobial Agents and Chemotherapy, vol. 49, no. 1, pp. 269–275, 2005. References [11] B. O. Schroeder, D. Ehmann, J. C. Precht et al., “Paneth cell α- defensin 6 (HD-6) is an antimicrobial peptide,” Mucosal [1] J. Neu and W. A. Walker, “Necrotizing enterocolitis,” The New Immunology, vol. 8, no. 3, pp. 661–671, 2015. England Journal of Medicine, vol. 364, no. 3, pp. 255–264, “ α 2011. [12] H. Chu, M. Pazgier, G. Jung et al., Human -defensin 6 pro- “ motes mucosal innate immunity through self-assembled pep- [2] R. Sharma, J. J. Tepas III, M. L. Hudak et al., Portal venous gas ” – and surgical outcome of neonatal necrotizing enterocolitis,” tide nanonets, Science, vol. 337, no. 6093, pp. 477 481, 2012. Journal of Pediatric Surgery, vol. 40, no. 2, pp. 371–376, 2005. [13] E. B. Mallow, A. Harris, N. Salzman et al., “Human enteric ” [3] D. Duro, L. A. Kalish, P. Johnston et al., “Risk factors for intes- defensins. Gene structure and developmental expression, – tinal failure in infants with necrotizing enterocolitis: a Glaser Journal of Biological Chemistry, vol. 271, no. 8, pp. 4038 Pediatric Research Network study,” The Journal of Pediatrics, 4045, 1996. vol. 157, no. 2, pp. 203–208.e1, 2010. [14] M. A. Underwood, “Paneth cells and necrotizing enterocoli- ” – [4] L. Berman and R. L. Moss, “Necrotizing enterocolitis: an tis, Gut Microbes, vol. 3, no. 6, pp. 562 565, 2012. update,” Seminars in Fetal & Neonatal Medicine, vol. 16, [15] F. H. Heida, G. Beyduz, M. L. C. Bulthuis et al., “Paneth no. 3, pp. 145–150, 2011. cells in the developing gut: when do they arise and when are Gastroenterology Research and Practice 7

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